ALBERT

Beschreibung: Introduction Non-Hodgkin lymphoma (NHL) is the most common hematologic malignancy and the fifth leading cause of cancer death in the world; over 90% are B cell lymphomas and express CD-20 surface antigen [1]. CD-20 antigen is a transmembrane protein of 33-37 kDa protein located on the surface of pre-B and mature lymphocytes B [2, 3]. It is expressed in 90% of B-cell NHL [4]. Anti-CD20 antibody (Rituximab®), a specific chimeric monoclonal antibody directed against CD20 on B lymphocytes and the first monoclonal antibody approved for the treatment of CD20+ NHL. The development of new agents directed against specific targets may improve the sensitivity and specificity of imaging for its staging. Objective To radiolabel Rituximab with 99mTc and evaluate its properties as a potential imaging agent for NHL. Methodology Rituximab was derivatized with succinimidyl-hydrazinonicotinamide (Suc-HYNIC) and MALDI TOF/TOF was used to confirm the level of HYNIC conjugation to Rituximab. This antibody was radiolabeled with 99mTc using a mixture of Tricine/SnCl2.2H2O. Radiochemical purity was determined by ITLC-SG, size exclusion chromatography and HPLC. In-vitro stability was studied in solution; serum and L-Cysteine up to 24 h. In-vitro binding and competition assays were performed with Ramos and Raji NHL cell lines up to 120 min and were analyzed by laser confocal microscopy. Ramos and Raji cells were incubated with buffer to evaluate any degree of autofluorescence. Biodistribution studies were performed in normal Balb/C mice at 1, 4, 24 and 48 h (n = 5). Results HYNIC- Rituximab was efficiently labeled with 99mTc. The in-vitro radiochemical stability studies of the radiolabeled antibody showed that the complexes formed were stable and no significant transchelation was detected. In-vitro binding and displacement assays confirm that after derivatization and labeling, Rituximab retained its specificity of binding to CD-20 antigen. Immunoreactivity of HYNIC-Rituximab to Ramos and Raji cell lines was determined by direct immunofluorescence. We observed a remarkable cell membrane staining of the NHL cells. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). After its incubation with buffer, auto fluorescence was discarded. These results show that Rituximab´s affinity for NHL cells remained unaffected after its derivatization. Biodistribution studies were performed to quantify localization and clearance of the radiolabeled antibody complex in normal tissues. Significant accumulation of radioactivity was found in liver, indicating that the primary route of clearance was hepatic. Other major uptake was not found after evaluating the rest of the organs at the observed time points. Conclusions 99mTc-HYNIC-Tocilizumab was easily and rapidly labeled, with radiochemical purities greater than 90%, retaining its specificity of binding. Our results indicate that 99mTc-HYNIC-Rituximab represents a promising molecular imaging agent for NHL. Acknowledgments ANII, Roche Laboratories, Pro.In.Bio. PEDECIBA Química References [1] Swerdlow AJ. Epidemiology of Hodgkin’s disease and nonHodgkin’s lymphoma. Eur J Nucl Med Mol Imaging 2003; 30 Suppl 1:S2–12. [2] Einfield DA, Beown JP, Valentine MA, et al. Molecular cloning of the human cell CD20 receptor predicts a hydrophobic protein with multiple transmembrane domains. EMBO J 1988; 7, 711-717. [3] Valentine MA, Meier KE, Rossie S, et al. Phosphotylation of the CD20 phosphoprotein in resting B lymphocytes. Regulation by protein kinase. C J Biol Chem 1989; 264, 11282-11287. [4] Tedder TF, Boyd AW, Freedman As, et al. The B cell surface molecule B1 is functionally linked withBcell activation and differentiation. J Immunol 1985; 135, 973-979. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Background Anti-multiple myeloma (MM) vaccinations have recently emerged as a strategy to eradicate MM cells in patients with residual tumor mass. However, most vaccination studies reported transient immune responses, involving CD8+ T-cells or antibodies responses in patients with predefined MHC repertoire. Signal peptide (SP) domains, a 13-50 amino acid long peptides, have been demonstrated to present an exceptionally high number of antigen specific MHC class I, class II and B-cell epitopes per sequence length. Therefore, they are suitable to induce a robust immune response combining CD8+, CD4+ T-cells and antibodies across HLA barrier. Following this rationale we developed ImMucin, A 21-mer therapeutic vaccine encoding the entire SP domain of the MUC1 tumor-associated antigen (TAA), which is over expressed by most carcinomas and hematological tumors including MM. Methods A phase I/II study to assess the safety (primary endpoint) and efficacy (secondary endpoint) of ImMucin was conducted in MM subjects with stable or progressive residual asymptomatic MUC1+ disease who have already had upfront auto-SCT. Patients recruited into the study initially received 6 bi-weekly vaccination of intra-dermal 100ug ImMucin plus 250ug hGM-CSF. Based on immune and clinical response eligible patient could receive additional 6 bi-weekly vaccination of 100ug or 250ug ImMucin plus 250ug hGM-CSF. Immunomonitoring analysis included; ImMucin-specific IFN-g production in CD4+ and CD8+ T-cells, HLA-A2.1 tetramer binding in CD8+ T-cells, proliferation and antibodies production. Positive response in these assays vs. pre vaccination levels were; x2 increase in IFN-g and 〉0.5%+ T-cells, any increase in tetramer binding levels, x2 increase and stimulation index (SI)〉2 and any increase in anti-ImMucin titer respectively. Results 15 patients, median age 58 years participated in the study. The median number of prior therapies was 2. Median time from diagnosis and from auto-SCT to vaccination were 25 months (12-143) and 15 months (3-134) respectively. 9 patients had post auto-SCT residual MM and 6 had an evidence for biochemical progression. 11 patients completed the vaccination program while 4 discontinued the program due to PD. ImMucin was shown to be safe with no vaccine related grade ≥3 adverse events (AEs). Patients had temporary, grade 1,2 AEs, including injection related rash (n=8), fatigue (n=6), weakness, self-resolving fever and muscle pain (n=5). IFN-g production response to ImMucin was highly positive in all patients with 4-80 folds increase for CD4+ and 18-80 folds increase for CD8+. Mean baseline and peak post vaccination levels for ImMucin-specific CD4+ T-cells were 0.21% and 4.07%, respectively (P

Beschreibung: Introduction Richter's syndrome (RS) is the rare development of an aggressive lymphoid malignancy in a patient with pre-existing, or concomitant chronic lymphocytic leukemia (CLL). This complication occurs in about 5 % of patients with CLL, and the most frequent form is the development of diffuse large B-cell lymphoma (DLBCL) and less frequently Hodgkin lymphoma (HL) or prolymphocytic leukemia (PLL). Most of the available data on RS is derived from case reports or small series of patients, and only a few larger cohorts have been published. Aim The purpose of this retrospective study was to summarize our experience with RS in CLL, examine possible risk factors and analyze relevant demographic, laboratory and clinical parameters, including outcome. Methods We collected a total of 119 patients diagnosed with RS from 12 medical centers in Israel during the period 1971-2010.We then summarized clinical, demographic and some biological features related to CLL at diagnosis and examined possible risk factors for their transformation to RS. Results Of the 119 patients with RS, 61 % were males, 82% developed DLBCL, 14% HL and 4% PLL. In terms of ethnicity: 95% were Jews (64% Ashkenazi) and only 5 % were Arabs, which is similar to the reported data for CLL in Israel. The median time from CLL diagnosis to development of RS was 60 months (range, 0-182 months), and the median overall survival from diagnosis of RS was 9.5 months;34 % of the cases developed extranodal RS, and the most frequent sites of involvement were the gastro-intestinal tract and bone marrow. These results further confirm that there are no established “sanctuary sites” of extranodal RS, which can develop in all tissues and organs. None of the conventional clinical and laboratory parameters examined and evaluated as possible risk factors in CLL were able to predict transformation to RS, including: occurrence of autoimmune phenomenon during the course of CLL, spleen size, serum beta 2 microglobulin levels, degree of CD38 positivity, and the number of previous treatments given for CLL, or the prior use of rituximab in these regimens. The only parameters, present at the time of RS diagnosis which were found to correlate with adverse prognosis, included: performance status〉2 (HR- 3.45), high IPI (HR- 3.28) and high Richter score (HR-7.45). In regard to therapy for RS, patients treated with chemotherapy followed by autologous stem cell transplantation (ASCT) had a better outcome with improved overall survival (p=0.034) (Figure). Conclusions RS remains a heterogeneous entity. In this large series of patients none of the conventional clinical, epidemiological or laboratory parameters, often evaluated as possible prognostic factors in CLL, were associated with the risk of transformation to RS. In this retrospective study molecular genetic data were not available to examine their possible significance, as reported recently in other series. In terms of therapy, prior treatment of CLL with rituximab – containing regimens did not appear to impact the eventual outcome of patients who developed RS. On the other hand, ASCT did significantly improve overall survival in patients who had transformation to RS. On behalf the Israeli CLL study Group. Disclosures: No relevant conflicts of interest to declare.